1. Field of the Invention
The present invention relates generally to leptin binding domain compositions and methods thereto. The compositions are useful for detecting the presence of leptin in a sample and distinguishing free leptin from bound leptin in multiple species. More specifically, the present invention relates to methods for determining free leptin in a sample from an individual by assaying a sample for the binding of leptin to an avian leptin receptor binding domain, and detecting the bound leptin by using a labeled anti-leptin antibody.
2. Description of the Related Art
Human leptin is a 16 kDa, 146 amino acid residue, non-glycosylated polypeptide that was described based on the genetic mapping of a recessive mutation that caused severe obesity in mice (1-2). Abolished transcription or production of an inactive obesity (ob) gene product was found to be responsible for producing the obese (ob/ob) mouse, which is characterized by severe obesity, hyperphagia, hyperglycemia, hyperinsulinemia and insulin resistance, hypothermia, and infertility (3-5). The obesity gene product, later known as leptin, is produced by the adipose tissue across a wide range of animal species (1, 2) and shows high interspecies conservation, with the human leptin sharing up to 84% and 87% homology with rat and mouse leptin, respectively (1, 6). Crossing the blood-brain barrier (7), circulating leptin is believed to convey vital information to the brain regarding the level of body fat and energy stores and activate the hypothalamic centers that regulate food intake and energy expenditure (3, 4, 7, 8). A number of recent reports have proposed adipose tissue as an important endocrine gland, and have identified leptin as a pleiotrophic hormone affecting many different organs and tissues in the body (9-11). In addition to regulating fat metabolism, involvement of leptin in pathophysiology of multiple endocrine feedback loops, including reproductive, hematopoiesis, and adrenal cortex function, as well as immune system function, have been reported (8, 9, 11-13).
The effects of leptin on various functions may be mediated centrally via changes in hypothalamic neuropeptide Y expression, which in turn regulates the secretion of gonadotropic hormones (36) and food intake (37). Metabolic changes induced by alterations in food intake affect various hormone systems indirectly. In addition to its systemic effects, direct peripheral leptin actions have been demonstrated in several target tissues. Thus, leptin has been shown to modulate insulin activity in hepatocytes in vitro (38). Leptin modulates ovarian steroidogenesis in vitro (39, 40) and affects angiogenesis, acting in some tissues as a positive angiogenic factor (41), whereas it is angiostatic in adipose tissues (42). In rat ovary, leptin attenuates apoptosis and thus enhances sexual maturation (43). Leptin also regulates several functions in the pituitary cells (44).
Leptin circulates in serum as a free form or bound to leptin-binding proteins, such as a soluble form of leptin-binding receptor (35). The majority of leptin is in the bound form in lean individuals, but in the free form in obese individuals (35). In addition, leptin levels are also influenced by the stage of puberty and gender in both adults and children. Blood leptin levels are about 2-3 fold higher in men than in women (3, 4, 15-18); its secretion is pulsatile (19) and follows a circadian rhythm, with the highest levels achieved during the night (19, 20). Obesity in man, in contrast to obesity in mice, is associated with a significant increase in circulating leptin levels (14). Fat mass is the main determinant of leptin to the extent that its circulating levels are exponentially correlated with body mass index (BMI) and percent body fat (2-4, 14). The high sensitivity of leptin to changes in body fat is responsible for the observed wide variations in plasma leptin, which could range from 0.03 to over 100 ng/mL (15-18). Studies have indicated that loss of body fat decreases serum leptin levels, while an increase in body fat increases leptin levels; these observations suggest that the body's adaptive response to low serum leptin differs from the response to high leptin levels in a way that is characteristic for obesity (35).
Recombinant leptins from several farm animals, such as sheep (45), chicken (46), cow, pig (47), and humans (48) have been prepared. A variety of in vivo experiments performed with leptin-deficient ob/ob and normal mice (49-51), as well as with chicken and sheep (52-54), indicate that administration of leptin by direct intraventricular, intramuscular, or intraperitoneal injections leads to a remarkable decrease in food intake and subsequent weight loss. The main target of leptin's action is located in the brain, and as leptin is produced in adipose tissue, it has to be transferred through the blood-brain barrier. This transfer is mediated mainly through the short form of the leptin receptor located in the choroid plexus (55, 56). The leptin receptor is a member of the cytokine family of receptors and is responsible for mediating the biological activity of leptin (3, 4). In humans, four different mRNA splice variants of the leptin receptor have been so far identified (3, 4). Accordingly, secreted leptin may circulate in both free (unbound) as well as in complex forms bound to a number of different binding proteins. The latter reportedly includes a soluble splice variant of the receptor that has no transmembrane domain as well as soluble leptin receptor generated by the proteolytic cleavage of the membrane-anchored receptors and possibly other unidentified leptin binding proteins (3, 4, 22-23). Although the nature as well as biological importance of leptin association with binding proteins has not been fully defined, recent evidence has proposed a distinct role for the free and bound leptin (24). In addition, leptin association with binding proteins is thought to increase leptin biovailability and half-life as well as possibly contributing to the state of leptin resistance (2, 3, 4, 19). As the balance of free and complex leptin is influenced by a complex array of variables, including several hormones and growth factors (3, 4), accurate determination of leptin sub-fractions could be of significant value in advancing our understanding of pathophysiology and potential diagnostics and therapeutic (10) applications of leptin.
Progress in leptin research was complemented by the discovery and cloning of the leptin receptor (3, 4, 21). While polynucleotides and polypeptides of chicken leptin receptor are reportedly provided in Horev, et al. (57) and WO 01/30963, the particular binding domain for the avian species was not provided. A citation of exons 9 and 10 by Fong (58) is in reference to the mammalian genes of humans and mice. WO 01/30963 reports only a 49-50% sequence similarity between those species and the chicken receptor amino acid sequences.
Continued progress in leptin research has also been aided by development of various methodologies for functional as well as clinical investigations of leptin (3, 4, 10). Currently, immunoassays are the method of choice for leptin quantification in serum and other biological fluids. The initial immunoassays, based on competitive principles (2, 25), have been largely replaced with non-competitive, non-isotopic, methodologies (26), which have the analytical performance advantages as well as avoiding the draw backs of radioactive labels. These immunoassays do not completely distinguish between the various circulating leptin forms (e.g., free vs complex, vs total) (26). Given the differential response of free and bound leptin to caloric intake, obesity, and other hormones as well as their potentially distinct pathophysiological roles (3, 4, 24), development of methodologies for specific determinations of free or complex leptin forms would be of significant value. In this context, a conventional, one-step, enzyme-linked immunosorbent assay (ELISA) for free leptin has been recently described (27). Development of a functional bioassay for free leptin has been also reported (28), but the methodology is relatively cumbersome and expensive for routine and large-scale applications.